Copper Glufosinate-Based Metal–Organic Framework as a Novel Multifunctional Agrochemical

Pesticides are agrochemical compounds used to kill pests (insects, rodents, fungi, or unwanted plants), which are key to meet the world food demand. Regrettably, some important issues associated with their widespread/extensive use (contamination, bioaccumulation, and development of pest resistances) demand a reduction in the amount of pesticide applied in crop protection. Among the novel technologies used to combat the deterioration of our environment, metal–organic frameworks (MOFs) have emerged as innovative and promising materials in agroindustry since they possess several features (high porosity, functionalizable cavities, ecofriendly composition, etc.) that make them excellent candidates for the controlled release of pesticides. Moving toward a sustainable development, in this work, we originally describe the use of pesticides as building blocks for the MOF construction, leading to a new type of agricultural applied MOFs (or AgroMOFs). Particularly, we have prepared a novel 2D-MOF (namely, GR-MOF-7) based on the herbicide glufosinate and the widely used antibacterial and fungicide Cu2+. GR-MOF-7 crystallizes attaining a monoclinic P21/c space group, and the asymmetric unit is composed of one independent Cu2+ ion and one molecule of the Glu2– ligand. Considering the significant antibacterial activity of Cu-based compounds in agriculture, the potential combined bactericidal and herbicidal effect of GR-MOF-7 was investigated. GR-MOF-7 shows an important antibacterial activity against Staphylococcus aureus and Escherichia coli (involved in agricultural animal infections), improving the results obtained with its individual or even physical mixed precursors [glufosinate and Cu(NO3)2]. It is also an effective pesticide against germination and plant growth of the weed Raphanus sativus, an invasive species in berries and vines crops, demonstrating that the construction of MOFs based on herbicide and antibacterial/antifungal units is a promising strategy to achieve multifunctional agrochemicals. To the best of our knowledge, this first report on the synthesis of an MOF based on agrochemicals (what we have named AgroMOF) opens new ways on the safe and efficient MOF application in agriculture.


Supporting information (SI). Details of selected bond lengths and angles are given in
. CCDC reference number for the structure is 2109530.

Water stability studies
UV-vis spectra were recorded to investigate the stability of GR-MOF-7 in water at pH = 6. Solutions were prepared via dilution of a 50 µM GR-MOF-7 stock. Further, the stability of GR-MOF-7 was confirmed by 1 H NMR, by dissolving a small amount of GR-MOF-7 and glufosinate linker (ca. 5 mg) in deuterated water for 24 h. The particle size and ζ-potential determinations were performed using a Malvern Nano-ZS, Zetasizer Nano series. Aroun 1 mg of GR-MOF-7 was dispersed in 1 mL of water using an ultrasound tip (10% amplitude, 30 s) for 1 min. Size and ζpotential were analyzed in triplicate (n = 3).
6 mg of GR-MOF-7 was loaded in a 1.27 mm polyimida capillary (95820-11; Cole-Parmer, Illinois, USA), humidified with 50 µL of distilled water, and mounted on a spinner rotating at about 5 Hz (V= 45 kV, I= 40 mA) to improve the particles' statistics. Diffraction data were collected each hour over a 24 h time period in a Bruker D8 Advanced equipment.

Antibacterial activity
The Gram-positive Staphylococcus aureus (CECT 240, strain designation ATCC 6538P) and Gramnegative Escherichia coli (CECT 516, strain designation ATCC 8739) bacteria were used as reference strains for the antibacterial activity tests. The microorganisms were preserved at −80ºC in glycerol (20% v/v) until their use. Reactivation was performed in nutrient broth (1 mL of inoculums in 20 mL of NB; composed by 5 g·L −1 beef extract, 10 g·L −1 peptone, 5 g·L −1 NaCl, pH=7-7.2) at 37 °C under stirring (100 rpm) and routinely tracked by measuring OD at 600 nm (Shimadzu UV-1800 spectrophotometer) to preserve the exponentially growing phase of the microorganisms during the total time of contact (20 h). Innocuous of 10 6 cells·mL -1 of both bacteria were prepared in 1/500 NB. For the antibacterial experiments, diverse concentrations of GR-MOF-7 solutions (0, 0.5, 1, 1.5, 2.5, 3.5, 4.5, 5, 25, 50, 100, 250 ppm) were prepared as well as control samples (glufosinate, Cu(NO 3 ) 3 ·3H 2 O, and glufosinate + Cu(NO 3 ) 3 ·3H 2 O), being dissolved over 2.4 mL of the previously mentioned 10 6 cells·mL -1 inoculums of a 24-well plate. In the case of the controls, for each desired concentration, the quantity of each constituent was adjusted to the corresponding part of the bulk MOF (e.g., 1 ppm of GR-MOF-7 corresponds to 0.65 ppm of glufosinate + 1 ppm of Cu(NO 3 ) 3 ·3H 2 O). After 20 h of incubation at 37ºC without stirring, the bacterial viability was evaluated by determining: i) colony-forming units (CFU), where bacteria aliquots were placed in sterile 96 well plates in 10fold serial dilutions in phosphate-buffered solution (PBS). Replicated 10 µL spots were placed on Petri dishes containing NB agar-medium and after 24 h incubation at 37 °C without stirring, CFU were counted using a CL-1110 counting instrument (Acequilabs, Spain). For colony number estimations, at least three replicates of at least two serial dilutions were considered in order to obtain an estimated inhibition (CFU . mL -1 ); and by ii) fluorescein diacetate staining (FDA), non-fluorescent compound hydrolyzed by esterases in fully functional cells to a green fluorescent compound (fluorescein), which is an extensively used indicator for S. aureus and E. coli enzymatic activity determination. Thus, the liquid fraction was analysed in 96-well black microplates by mixing 5 µL of FDA (2 mg·mL -1 in dimethyl sulfoxide, DMSO) with 195 µL of bacterial suspension in each well. The plate was incubated at 25 ºC for 30 min, with continuous readings every 5 min (λ ex = 485 nm; λ em = 538 nm) using a fluorometer (Fluoroskan Ascent FL Fluorimeter/Luminometer; Thermo Scientific, Waltham, MA, USA). The possible fluorescence interference of the culture medium and GR-MOF-7 was also checked. 4 Each sample was measured for quadruple, disclosing the outcomes as an inhibition percentage, calculated as the difference in fluorescent intensity of the sample with respect to one of the control blank assays. Moreover, the fluorometer was also used for the determination of the bacterial oxidation «ROS production». 5,6 Briefly, 150 µL of each sample fraction was incubated for 30 min in 96-well black microplates with 50 µL of 10 mol . L -1 of the ROS salt (2´,7´dichlorodihydrofluorescein diacetate, H 2 DCF-DA), which is sensitive for hydrogen peroxide and other oxidative species, including hydroxyl and peroxyl radicals. Each sample was measured for quadruple readings, every 5 min (λ ex = 495 nm; λ em = 525 nm) and represented normalized with respect to the negative control group for a direct comparison.
Confocal laser scanning microscopy (CLSM) was performed for visual and qualitative assessment of antibacterial activities. Cell images of each bacteria strain were obtained after the contact with the GR-MOF-7 suspensions (50 µL aliquot) via confocal microscopy using a Confocal SP5 (Leica Microsystems, Germany). The bacteria were stained with a LIVE/DEAD kit (Live/Dead BacLight Viability Kit, Thermo Fisher, USA), which consists in a fluorescent dye, prepared with a mixture of Propodium iodide (PI) and Syto 9 in DMSO (10 µL of each dye and 980 µL of DMSO). This staining permeates the cell membrane depending on its integrity: viable cells exhibit green fluorescence (Syto 9: live cells, λ ex = 480 nm; λ em = 500 nm), whereas non-viable bacteria provide red intensity (PI: dead cells, λ ex = 490 nm; λ em = 635 nm). The fluorescent dye mixture (10 µL) incubation in contact with the different bacteria took place during 30 min in dark at RT.

Effect on seeds germination
Seeds of Rabanus satibus (radish) were purchased from authorized dealer with certification ES-ECO-001-AN European Agriculture Ecojaral -Productos Ecológicos, Granada, Spain (www.ecojaral.com). The average germination rates of plant seed were greater than 90%. Seeds were kept in a dry place in the dark under room temperature before use.
Previous to any test, the active concentration of glufosinate ammonium against R. sativus, used in this study as model invasive weed, was determined. The concentration was selected according to the recommendations of the commercial glufosinate based pesticide BASF-Rely280®. 7 Aqueous solutions (5 mL) of glufosinate ammonium with different concentrations (R. sativus: 6.35·10 -3 , 0.012 and 0.025 M) were tested. In parallel, a water control was also performed. 7 cm 2 Petri dishes were used for each different concentration with a total of 20 seeds per petri dish. Radish seed germination was studied for 12 days. During this time, control seeds develop different growth stages (1, 2, 3 and 4), as shown in Figure S1 The number of germinated seeds was counted to obtain the germination rate (GR).

GR = seeds germinated/total seeds x 100
Once the active concentration of glufosinate ammonium was determined, the activity of GR-MOF-7 was studied against R. sativus. 7 mL of an aqueous solution of GR-MOF-7 (2.55 g·L -1 or 0.010 M) were added to a petri dishes containing 20 weeds following the same procedure as previously described. In parallel, three different controls (water, glufosinate, and glufosinate + Cu(NO 3 ) 2 ·3H 2 O) were performed using the same concentration (0.010 M). All tests were carried out at least in triplicate.

Effect on pest (seed and plant) growth
Experiments were conducted in Granada (Spain) under ambient conditions, with an average day/night photoperiod of 15/10 h, and temperature 30/15 ºC and in a dry environment. R. sativus germinated seeds were sown in 15 x 10 cm flowerpots containing artificial mixed soil (Compo Sana), and each pot contained 10 plants. R. sativus plants were watered daily to optimize their growth. When the plants reach the 3-leaf stage, they were sprayed once with 1 mL of GR-MOF-7 and glufosinate ammonium solution (0.010 M) using a microaerosol sprayer. Water was used as control. All tests were carried out at least in triplicate.

Effect on non-target plant
Experiments were conducted in Granada (Spain) under ambient conditions, with an average day/night photoperiod of 15/10 h, and temperature 40/15 ºC and in a dry environment. Ribes nigrum (berry) plants were sprayed once with 1 mL of GR-MOF-7 solution (0.010 M) using a microaerosol sprayer.

Statistics
The results of the different assays are represented as mean ± standard deviation (SD). Ordinary Two-way ANOVA analysis of variance followed by a Tukey´s multiple comparison tests were carried out to determine significant differences using GraphPad Prism 9.2 software (GraphPad Software, Inc., La Jolla, CA, USA). Each experiment was performed at least three times (n  3). In the graphs, the results are indicated as: P > 0.05, *P  0.05, **P  0.01, ***P  0.001 and ****P  0.0001.  Table S2. Selected bond lengths (Å) and angles (°) for GR-MOF-7.

Powder XRD refinement of GR-MOF-7
The lattice parameters were refined using TOPAS software (version 5, Bruker AXS, Karlsruhe, Germany). There is a good agreement between the data and the model.